Receptor-gated Ion Channels Research Articles

Genetic Mutation of GluN2B Protects Brain Cells Against Stroke Damages.

Immediately following ischemia, glutamate accumulates in the extracellular space and results in extensive stimulation of its receptors including N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors. A large amount of Ca2+ influx directly through the receptor-gated ion channels which leads to Ca2+ overload and triggers several downstream lethal reactions. As a result, cell dies via apoptosis or necrosis, or both. Death-associated protein kinase 1 (DAPK1) physically and functionally interacts with the NMDA receptor GluN2B subunit at extra-synaptic sites and this interaction acts as a central mediator for stroke damage. The goal of this study is to explore an effective strategy in the treatment of stroke with a molecular genetic manipulation to interrupt DAPK1-GluN2B interaction. We generated a mutant strain of mice with the conditional deletion of GluN2B C-terminal tail consisting of amino acids 886-1269 in the forebrain excitatory neurons (the GluN2B mutant mice) and tested the protective effects of this mutation in stroke damages. GluN2B mutation effectively disrupted the DAPK1-GluN2B interaction and inhibited extra-synaptic NMDA receptor currents without affecting synaptic NMDA receptor channel activity in the central neurons. GluN2B mutation protected against stroke damages both in vitro and in vivo and hence improved behavioral performance. Disruption of the DAPK1-GluN2B interaction is therapeutically effective against stroke damages.

CXCL12-induced neurotoxicity critically depends on NMDA receptor-gated and L-type Ca2+ channels upstream of p38 MAPK.

BackgroundThe chemokine receptor CXCR4 (CD184) and its natural ligand CXCL12 contribute to many physiological processes, including decisions about cell death and survival in the central nervous system. In addition, CXCR4 is a co-receptor for human immunodeficiency virus (HIV)-1 and mediates the neurotoxicity of the viral envelope protein gp120. However, we previously observed that CXCL12 also causes toxicity in cerebrocortical neurons but the cellular mechanism remained incompletely defined.MethodsPrimary neuronal-glial cerebrocortical cell cultures from rat were exposed to a neurotoxicity-inducing CXCL12 concentration for different times and the activity of the stress-associated mitogen-activated protein kinase p38 (p38 MAPK) was assessed using an in vitro kinase assay. Neurotoxicity of CXCL12 and cellular localization of p38 MAPK was analyzed by immunofluorescence microscopy. Pharmacological inhibition of NMDA-type glutamate receptor-gated ion channels (NMDAR) of l-type Ca2+ channels was employed during 12- and 24-h exposure to neurotoxic amounts of CXCL12 to study the effects on active p38 MAPK and neuronal survival by Western blotting and microscopy, respectively. Neurotoxicity of CXCL12 was also assessed during pharmacological inhibition of p38 MAPK.ResultsHere, we show that a neurotoxic amount of CXCL12 triggers a significant increase of endogenous p38 MAPK activity in cerebrocortical cells. Immunofluorescence and Western blotting experiments with mixed neuronal-glial and neuron-depleted glial cerebrocortical cells revealed that the majority of active/phosphorylated p38 MAPK was located in neurons. Blockade of NMDAR-gated ion channels or l-type Ca2+ channels both abrogated an increase of active p38 MAPK and toxicity of CXCL12 in cerebrocortical neurons. Inhibition of l-type Ca2+ channels with nimodipine kept the active kinase at levels not significantly different from baseline while blocking NMDAR with MK-801 strongly reduced phosphorylated p38 MAPK below baseline. Finally, we confirmed that directly blocking p38 MAPK also abrogated neurotoxicity of CXCL12.ConclusionsOur findings link CXCL12-induced neuronal death to the regulation of NMDAR-gated ion channels and l-type Ca2+ channels upstream of p38 MAPK activation.

+]-Huperzine A treatment protects against N-methyl- d-aspartate-induced seizure/ status epilepticus in rats

The toxicity of organophosphorous (OP) nerve agents is attributed to their irreversible inhibition of acetylcholinesterase (AChE), which leads to excessive accumulation of acetylcholine (ACh) and is followed by the release of excitatory amino acids (EAA). EAAs sustain seizure activity and induce neuropathology due to over-stimulation of N-methyl- d-aspartate (NMDA) receptors. Huperzine A (Hup A), a blood–brain barrier permeable selective reversible inhibitor of AChE, has been shown to reduce EAA-induced cell death by interfering with glutamate receptor-gated ion channels in primary neuronal cultures. Although [−]-Hup A, the natural isomer, inhibits AChE approximately 38-fold more potently than [+]-Hup A, both [−]- and [+]-Hup A block the NMDA channel similarly. Here, we evaluated the protective efficacy of [+]-Hup A for NMDA-induced seizure in a rat model. Rats implanted with radiotelemetry probes to record electroencephalography (EEG), electrocardiography (ECG), body temperature, and physical activity were administered various doses of [+]-Hup A (intramuscularly) and treated with 20 μg/kg NMDA (intracerebroventricular) 20–30 min later. For post-exposure, rats were treated with [+]-Hup A (3 mg/kg, intramuscularly) 1 min after NMDA (20 μg/kg). Our data showed that pre- and post-exposure, [+]-Hup A (3 mg/kg) protects animals against NMDA-induced seizures. Also, NMDA-administered animals showed increased survival following [+]-Hup A treatment. [+]-Hup A has no visible effect on EEG, heart-rate, body temperature, or physical activity, indicating a reduced risk of side effects, toxicity, or associated pathology. Our results suggest that [+]-Hup A protects against seizure and status epilepticus (SE) by blocking NMDA-induced excitotoxicity in vivo. We propose that [+]-Hup A, or a unique combination of [+]- and [−]-Hup A, may prove to be effective for pre- and post-exposure treatment of lethal doses of OP-induced neurotoxicity.

Ca2+ Inhibits the Association of Memantine with N-Methyl-D-aspartate (NMDA) Receptor-Gated Ion Channels

We examined the effect of 1-amino-3,5-dimethyladamantane (memantine) and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) on the inhibition of [(3)H]MK-801 binding to crude synaptic membranes of rat forebrains in the absence or presence of Ca(2+). Ca(2+) decreased the potency of memantine to inhibit [(3)H]MK-801 binding. The effect of Ca(2+) was apparently competitive with memantine and was not annulled by the addition of Mg(2+). Ca(2+) slightly enhanced [(3)H]MK-801 binding, but showed no effect on the displacement of [(3)H]MK-801 binding by MK-801. The Ca(2+)-sensitive interaction of memantine with N-methyl-D-aspartate (NMDA) receptor-gated ion channels may provide a clue to understanding its voltage-dependent and clinically tolerated character.

New Roles for Interleukin-1 Beta in the Mechanisms of Epilepsy

The mechanisms that transform a normal brain to an epileptic one are not fully understood. Interleukin-1 beta (IL-1beta) contributes to neuronal degeneration observed in several neurological disorders and recently has been implicated in neuronal injury that may accompany the process of epileptogenesis. This review presents the hypothesis that IL-1beta may contribute to the development of epilepsy via several mechanisms, including classical effects on neuronal survival and transcription pathways; novel rapid effects on receptor-gated ion channels; and long-lasting effects on expression of selective gene families. Thus, evidence that IL-1beta actions in epilepsy can be independent from the neurotoxic effects of this cytokine is presented.

Repeated ethanol treatment in adolescent rats alters cortical NMDA receptor

Earlier we have reported that repeated ethanol treatment during adolescence causes long-lasting impairments in spatial learning and memory. The present study was undertaken to determine the cellular mechanisms underlying the persistent ethanol-induced cognitive dysfunction in adolescent male rats. Since in adult animals ethanol is known to affect the N-methyl- d-aspartate (NMDA) receptor–gated ion channel, the hypothesis tested here was that adolescent ethanol exposure modulates NMDA receptor (NR) regulation in the brain. Adolescent male rats were injected daily with ethanol (2 g/kg intraperitoneally) for 5 consecutive days. Control rats received isovolumetric saline for the same number of days. Groups of control and experimental rats were sacrificed 7 days after the last ethanol/saline administration, and NR activity was measured in specific brain regions (frontal cortex, hippocampus) using the [ 3H]MK-801 binding assay. In addition, some rats were sacrificed and their brains were used to investigate changes in NR pharmacology by measuring specific NR2 subunits immunohistochemically. Compared to saline-treated controls, ethanol-treated rats showed significant increases in [ 3H]MK-801 maximal binding in the frontal cortex. This was associated with increased cortical NR2B subunit protein. [ 3H]MK-801 binding in the hippocampus was minimally affected. These results indicate that ethanol exposure during the adolescent period produces brain region–specific alterations in NR activity. These changes are different from those reported in literature for ethanol administration during the perinatal period or adulthood. Together, these data suggest that adolescence represents a unique stage in brain development in its long-term sensitivity to ethanol.

Changes in magnesium concentration in the serum and cerebrospinal fluid of neuropathic rats

Central sensitization of neuropathic pain is associated with an influx of extracellular calcium via the opening of N-methyl-D-aspartate (NMDA) receptor-gated ion channels, which are usually blocked by magnesium plugs. As magnesium-deficient rats develop a mechanical hyperalgesia and intrathecal or intraperitoneal magnesium suppresses neuropathic pain, the magnesium concentrations in serum and cerebrospinal fluid may be altered in neuropathic pain. We therefore compared the magnesium concentrations in serum and cerebrospinal fluid of neuropathic rats with those in injured rats without symptoms of neuropathic pain and normal rats. Mechanical allodynia was induced in male Sprague-Dawley rats by tight ligature of the left lumbar fifth and sixth spinal nerves. The threshold of paw withdrawal was evaluated by the up-down method using withdrawal response to stimulus with a von Frey filament on the third, seventh and 14th days. Rats with a threshold of less than 4 g were selected as the symptomatic group and compared with an asymptomatic group, an unoperated control group and a sham-operated group. On the 16th day, the Mg2+ concentrations in serum and cerebrospinal fluid were measured. The magnesium concentrations in the serum and cerebrospinal fluid of symptomatic neuropathic rats did not differ from those in the injured rats without symptoms of neuropathic pain, sham-operated rats and normal rats. Our results suggest that physiologic homeostasis is maintained by active transport through the blood-brain barrier despite the activation of NMDA receptor-gated ion channels. However, rats with neuropathic pain may be in a magnesium-deficient condition at the effector site, such that magnesium treatment can decrease neuropathic pain.

Inhibition of Transient Lower Esophageal Sphincter Relaxation and Gastroesophageal Reflux by Metabotropic Glutamate Receptor Ligands

Transient lower esophageal sphincter relaxation (TLESR) is the major mechanism of gastroesophageal acid reflux. TLESR is mediated via vagal pathways, which may be modulated by metabotropic glutamate receptors (mGluRs). Group I mGluRs (mGluR1 and 5) have excitatory effects on neurons, whereas group II (mGluR2 and 3) and group III (mGluR4, 6, 7, and 8) are inhibitory. This study determined the effect of mGluRs on triggering of TLESR and reflux in an established conscious ferret model. Esophageal manometric/pH studies were performed in ferrets with chronic esophagostomies. TLESR were induced by a gastric load of 25 mL glucose (pH 3.5) and 30 mL air. In control treated animals, gastric load induced 3.52 +/- 0.46 TLESRs per 47-minute study, 89.7% of which were associated with reflux episodes (n = 16). The mGluR5 antagonist MPEP inhibited TLESR dose dependently, with maximal 71% +/- 7% inhibition at 35 micromol/kg (n = 9; P < .0001). MPEP also significantly reduced reflux episodes (P < .001) and increased basal lower esophageal sphincter pressure (P < .05). MPEP inhibited swallowing dose dependently, suggesting a common action on trigger mechanisms for swallowing and TLESR. The more selective analogue, MTEP, had more potent effects (90% +/- 6% inhibition TLESR at 40 micromol/kg; n = 8; P < .0001). In contrast, the group I agonist DHPG tended to increase TLESR. The group II agonist (2R, 4R)-APDC was ineffective, whereas the group III agonist L-(AP4 slightly reduced TLESR (33% at 11 micromol/kg; P < .05). The selective mGluR8 agonist (S)-3, 4-DCPG inhibited TLESR by 54% at 15 micromol/kg (P < .01). mGluR5 antagonists potently inhibit TLESR and reflux in ferrets, implicating mGluR5 in the mechanism of TLESR. mGluR5 antagonists are therefore promising as therapy for patients with GERD.

Modulation of synaptic zinc in barrel cortex by whisker stimulation

The cortical representation of the body surface is not fixed, but rather, is continuously modified by ongoing changes in sensory experience. Although the cellular and molecular mechanisms that subserve these changes are uncertain, increasing evidence suggests that synaptically-released zinc may play a role. Zinc is released from a subset of glutamatergic neurons and can modulate postsynaptic excitability by regulating the activation of glutamate and GABA receptor-gated ion channels. Previously, we have shown that whisker plucking, a manipulation commonly used to induce cortical map plasticity, results in a rapid and robust increase in staining levels for synaptic zinc in deprived regions of the barrel cortex. In the present study, we examined the effect of increased whisker activity, analogous to what may happen during tactile learning or exploratory behavior in a natural setting, on synaptic zinc levels in the adult barrel cortex. Our results indicate that stimulation of whiskers caused a selective decrease in zinc levels within layer 4 of the barrel hollow corresponding to the stimulated whisker. Quantitatively, levels of staining were significantly reduced at 3 h, and showed even greater reductions following 12 and 24 h of stimulation. However, these changes were not long-lasting, as levels of staining in the stimulated barrel returned to control values within 24 h after stimulation had ceased. These data indicate that zincergic circuits are highly sensitive to ongoing changes in sensory experience and may participate in moment-to-moment changes in the functional connectivity of the cerebral cortex.

P2X receptor activation elicits transporter-mediated noradrenaline release from rat hippocampal slices.

This study was designed to test the hypothesis of whether activation of presynaptic P2X receptor-gated ion channels elicits noradrenaline release from central catecholaminergic terminals. ATP, alpha,beta-methylene-adenosine 5'-triphosphate (alpha,beta-methyleneATP), and ADP elicited concentration-dependent [3H]noradrenaline outflow from superfused rat hippocampal slices with the following rank order of agonist potency: alpha,beta-methyleneATP > ATP > ADP. Among P2 receptor antagonists, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (30 microM), 4,4',4",4"'-[carbonylbis(imino-5,1,3-benzenetriyl-bis(carbonylimino))]tetrakis-1,3-benzenedisulfonic acid (100 nM), and 8,8'-[carbonybis(imino-3,1-phenylenecarbonylimino)]bis1,3,5-naphthalenetrisulphonic acid (10 microM) significantly inhibited the outflow of [3H]noradrenaline, evoked by ATP, whereas Brilliant Blue G (100 nM), 2'-deoxy-N6-methyladenosine 3',5'-bisphosphate tetraammonium (10 microM), the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (250 nM), and the A2A receptor antagonist 3,7-dimethyl-1-propargylxanthine (250 nM) were ineffective. Pretreatment with the Gi protein inhibitor pertussis toxin (2.5 microg/ml) did not change the effect of ATP on [3H]noradrenaline outflow. In contrast, a decrease in extracellular pH from 7.4 to 6.6 significantly attenuated the response by ATP. When extracellular Na+ was replaced by choline chloride and in the presence of the noradrenaline uptake inhibitor desipramine (10 microM), the ATP-evoked [3H]noradrenaline outflow was almost completely abolished, indicating that its underlying mechanism is the sodium-dependent reversal of the noradrenaline transporter. Reverse transcription-polymerase chain reaction analysis revealed that mRNA encoding P2X1, P2X2, P2X3, P2X4, P2X6, P2X7 and P2Y1 receptor subunits were expressed in the brainstem containing catecholaminergic nuclei projecting to the hippocampus, whereas mRNA encoding P2X5, P2Y2, P2Y4, and P2Y6 receptors were absent. Taken together, these results indicate that noradrenergic terminals of the rat hippocampus are equipped with presynaptic facilitatory P2X receptors, displaying a pharmacological profile similar to homomeric P2X1 and P2X3 receptors.

The effect of CGX-1007 and CI-1041, novel NMDA receptor antagonists, on NMDA receptor-mediated EPSCs

The N-methyl- d-aspartate (NMDA) receptor-gated ion channel is comprised of at least one NR1 subunit and any of four NR2 subunits (NR2A–D). The NR2 subunit confers different pharmacological and kinetic properties to the receptor. CGX-1007 (Conantokin G), a 17-amino acid polypeptide isolated from the venom of Conus geographus, is a novel NMDA receptor antagonist that is thought to be selective for the NR2B subunit. CGX-1007 has been reported to have highly potent, broad-spectrum anticonvulsant activity in animal seizure models. CI-1041 is an investigational compound, which also possesses anticonvulsant activity and has been shown to be highly selective for NR2B containing NMDA receptors. Although both CI-1041 and CGX-1007 are reportedly NR2B specific antagonists, they differ in their ability to block amygdala-kindled seizures, suggesting that the mechanism of action of these compounds differs. The present study was designed to test the hypothesis that CI-1041 and CGX-1007 would differentially modulate the function of NMDA receptors at excitatory synapses. Using the whole cell patch clamp technique, CGX-1007 and CI-1041 were found to block CA1 pyramidal cell, NMDA receptor-mediated excitatory postsynaptic currents (N-EPSCs) in a concentration-dependent manner in hippocampal slices from P4–P6 animals. In contrast, only CGX-1007 decreased NMDA receptor-mediated EPSC peak amplitude in slices from adult animals. The CGX-1007 block of peak amplitude was accompanied by a similar concentration-dependent decrease in decay kinetics of NMDA receptor-mediated EPSCs. These results suggest that while CI-1041 may be selective for NMDA receptors containing the NR2B subunit, CGX-1007 appears to be less selective than previously reported.

The Changes in Serum and Cerebrospinal Fluid Ca2+, Mg2+, and Ca2+/Mg2+ Ratio in Neuropathic Rats

Background: Central sensitization of neuropathic pain is associated with an influx of extracellular calcium via the opening of N-methyl-D-aspartate (NMDA) receptor-gated ion channels, which is usually blocked by a magnesium plug. Many studies have found that intrathecal or intraperitoneal magnesium suppress neuropathic pain. Therefore, it is possible that serum and cerebrospinal fluid , , and / ratio are changed in neuropathic pain. The purpose of this study was evaluated changes in serum and cerebrospinal fluid , , and / ratio in neuropathic rats. Methods: Male Sprague-Dawley rats were prepared with tight ligation of the left lumbar 5th and 6th spinal nerves to produce neuropathic pain. The threshold of mechanical allodynia was evaluated by the up-down method using withdrawal response to a von Frey filaments stimulus on the 3rd, 7th, and 14th day. Rats with a threshold of less than 4 gram were selected as the experimental group. On the 16th day, serum and cerebrospinal fluid , , and / ratio were measured. Experimental group data were then compared with those of an unoperated control group and an unligated sham group; each group contained 10 animals. Results: No statistic differences were found between groups. Conclusions: Our results suggest that serum and cerebrospinal fluid , , and / ratio in neuropathic rats do not differ from those of normal rats because of physiologic homeostasis is maintained by active transport through the blood-brain-barrier despite of activation of NMDA receptor-gated ion channels. However, we believe that the ion-dependent voltage-gating in rats with neuropathic pain may be deficient in a chronic condition due to a decreased binding affinity of the NMDA receptor-gated channel, as has been found in hippocampal granule cells in epileptic rats.

Second International Symposium “Smooth Muscle Physiology and Biophysics”

The 2nd International Symposium on Smooth Muscle Physiology and Biophysics is organized jointly by the Bogomolets Institute of Physiology of the National Academy of Sciences of Ukraine (Kyiv, Ukraine), St. George’s Hospital Medical School of London University (Great Britain), and Taras Shevchenko National Kyiv University with support from the National Academy of Sciences of Ukraine, All-Ukraine Public Organization “Association of Pharmacologists of Ukraine,” The Wellcome Trust (Great Britain), and The Physiological Society (Great Britain). It will be held at the Bogomolets Institute of Physiology on October 28-31, 2003. The 1st Kyiv symposium “Smooth Muscle Physiology” was carried out 30 years ago in the tense international atmosphere of the cold war. However, many leading scientists were able to come from abroad, and the meeting seemed to open a window for cooperation between scientists from the USSR and their western colleagues. Today, the situation is entirely different, as many international research projects are conducted at the Bogomolets Institute, while Ukrainian scientists are performing research at universities throughout the world. The symposium will include lectures presented by prominent scientists from many countries on a wide range of smooth muscle physiology, biophysics, and pharmacology topics, including ionic mechanisms of electrical excitation, molecular organization, and biophysical properties of ion channels, neural regulation of smooth muscle motility, receptor-gated ion channels, mechanisms of action of neurotransmitters, calcium signals and active ion transport, excitationcontraction coupling, and mechanics of smooth muscle contraction. Twenty-five invited talks and short communications will be presented in seven sessions, namely: “Molecular Components of Cationic Channels: TRP and SOC,” “Neuronal Functions and Neurotransmitters,” “Endothelium and Smooth Muscle Function,” “Spontaneous Activity in Smooth Muscles,” “Responses of Smooth Muscle to Neurotransmitters,” “Regulation of Smooth Muscle Contraction,” and “Emerging Roles of Non-Smooth Muscle Cells in Smooth Muscle Tissues.” Two poster sessions, which will include about 50 research communications, are also planned. The program of the symposium will, thus, reflect the diversity of problems, techniques, and achievements of modern smooth muscle physiology and biophysics. We are hoping that the meeting will take place in a friendly and stimulating atmosphere of productive scientific exchange, which will enable young researchers to discuss problems and questions of modern smooth muscle physiology and biophysics with distinguished experts in this field. The Organizing Committee is happy to announce that the present symposium will coincide with the 75th birthday of Professor Mykhailo F. Shuba, who is one of the founders of contemporary smooth muscle physiology. We congratulate him on this event and wish Prof. Shuba good health and successful continuation of his important research.

Cyclic AMP-mediated regulation of striatal glutamate release: interactions of presynaptic ligand- and voltage-gated ion channels and G-protein-coupled receptors

The presynaptic regulation of striatal glutamate transmission was investigated using d-[3H]aspartate and mouse striatal slices. Functional changes in voltage-dependent and glutamate receptor-gated ion channels were elicited by pharmacologically modifying intracellular cyclic AMP formation via G-protein-coupled receptor stimulation. The kainate (KA)-evoked release was potentiated by the stimulatory G-protein (Gs)-coupled β-adrenoceptor agonist isoproterenol (ISO) in a concentration-dependent manner. This effect was mimicked by the specific calmodulin (CaM) antagonists trifluoperazine and calmidazolium. Tetrodotoxin (TTX), a blocker of Na+ channels, did not affect the basal release but inhibited to the same degree the releases evoked by kainate alone and by kainate and isoproterenol together. Vinpocetine, a blocker of voltage-dependent Na+ channels, did not alter the basal or the evoked release. The Na+ channel activator veratridine enhanced the basal release in a concentration-dependent manner and isoproterenol attenuated this effect. The opposite effects of isoproterenol on the kainate- and veratridine-evoked releases may reflect prevention of the cyclic AMP-protein kinase A (PKA) phosphorylation cascade in striatal glutamatergic signal transduction. In addition, the calmidazolium-induced potentiation of kainate-evoked release was thwarted by LY354740 and l-2-amino-4-phosphonobutanoate, agonists of the inhibitory G-protein (Gi)-coupled metabotropic group II and III glutamate receptors (mGluRs). Vinpocetine, which inhibits the CaM-dependent phosphodiesterase (PDE1), was likewise inhibitory. In turn, selective agonists and antagonists of the Gq-protein-coupled group I mGluRs and (S)-3,5-dihydroxyphenylglycine (3,5-DHPG) and (RS)-1-aminoindan-1,5-dicarboxylate (AIDA), which modulate the intracellular Ca2+ levels, did not alter the kainate-evoked release.The β-adrenoceptor-mediated cyclic AMP accumulation seems to downregulate Na+ channels but to enhance glutamate release by means of upregulation of kainate receptors. This regulation of presynaptic ligand- and voltage-gated ion channels is affected by the cAMP-protein kinase A-dependent phosphorylation cascade and controlled by Gi-protein-coupled mGluRs.

Stimulation of bitterness by capsaicin and menthol: differences between lingual areas innervated by the glossopharyngeal and chorda tympani nerves.

Capsaicin is viewed as a purely chemesthetic stimulus that selectively stimulates the somatosensory system. Here we show that when applied to small areas of the tongue, capsaicin can produce a bitter taste as well as sensory irritation. In experiment 1, individuals were screened for the ability to perceive bitterness from capsaicin on the circumvallate papillae. Fifteen of 25 subjects who reported at least weak bitterness rated the intensity of taste, irritation and coolness produced by 100-320 microM capsaicin and 100-320 mM menthol applied via cotton swabs to the tip (fungiform region), the posterior edge (foliate region), and the dorsal posterior surface (circumvallate region) of the tongue. Sucrose, citric acid, sodium chloride and quinine hydrochloride were applied to the same areas to assess tastes responsiveness. On average, capsaicin and menthol produced "moderate" bitterness (and no other significant taste qualities) in the circumvallate region, and weaker bitterness on the side and tip of the tongue. Sensory irritation from capsaicin was rated significantly higher at the tongue tip, whereas menthol coolness was rated higher in the circumvallate region. In experiment 2 we applied sucrose and quinine hydrochloride together with capsaicin to investigate the effects other taste stimuli might have on capsaicin's reported bitterness. As expected, adding quinine produced stronger bitterness in the circumvallate and fungiform regions, and adding sucrose significantly reduced the bitterness of capsaicin in the circumvallate region. Overall, the results suggest that capsaicin and menthol are capable of stimulating a subset of taste neurons that respond to bitter substances, perhaps via receptor-gated ion channels like those recently found in capsaicin- and menthol-sensitive trigeminal ganglion neurons, and that the glossopharyngeal nerve may contain more such neurons than the chorda tympani nerve. That some people fail to perceive bitterness from capsaicin further implies that the incidence of capsaicin-sensitive taste neurons varies across people as well as between gustatory nerves.

Local regulation of [3 H]-noradrenaline release from the isolated guinea-pig right atrium by P2X -receptors located on axon terminals

In this study the regulation of cardiac sympathetic outflow by presynaptic P(2X) receptor-gated ion channels was examined. ATP (30 microM - 1 mM) and other P2-receptor agonists elicited [(3)H]-noradrenaline ([(3)H]-NA) outflow from the isolated guinea-pig right atrium with the potency order of ATP>2-methyl-thioATP>alpha,beta-methylene-ATP=ADP, whereas ss, gamma-methylene-L-ATP was inactive. Ca(2+)-free conditions abolished both electrical field stimulation (EFS)- and ATP-evoked release of tritium. Unlike from EFS-induced outflow, ATP-induced [(3)H]-NA outflow was not reduced by omega-Conotoxin-GVIA (100 nM), Cd(2+) (100 microM) and tetrodotoxin (1 microM). The rapid extracellular decomposition of ATP was revealed by HPLC analysis. However, the effect of ATP to promote [(3)H]-NA release was not prevented by 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 250 nM), 3, 7-dimethyl-1-propargylxanthine (DMPX, 250 nM), or by reactive blue 2 (RB2, 10 microM), antagonists of A(1)-, A(2)- and inhibitory P(2) receptors. Zn(2+) (50 microM), the P(2X)-receptor modulator potentiated, and P(2X) receptor antagonists, i.e. suramin (300 microM), pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 30 microM) and 2'-o-(trinitrophenyl)-adenosine 5'-triphosphate (TNP-ATP, 30 microM) antagonized the ATP (1 mM)-evoked response. RT - PCR study revealed the expression of P(2X2) and P(2X3) receptor mRNAs in guinea-pig superior cervical ganglion. PPADS (30 microM) significantly reduced the EFS-induced [(3)H]-NA outflow in the presence DPCPX (250 nM) and RB2 (10 microM). In summary a P(2X)-type purinoceptor regulates noradrenaline release from the isolated right atrium of the guinea-pig. The pharmacological profile of the receptor resemble to homo-oligomeric P(2X3) or hetero-oligomeric P(2X2)/P(2X3) complexes, and provide a new target to intervene on sympathetic neuroeffector transmission at the presynaptic site.

Glutamate Receptors in the Cingulate Cortex, Hippocampus, and Cerebellar Vermis of Alcoholics

This study tests the hypothesis that glutamate receptors are altered in the brains of alcoholics as a result of chronic alcohol neurotoxicity. Excessive release of the excitatory neurotransmitter glutamate may damage postsynaptic neurons by increasing calcium flux through N-methyl-D-aspartate (NMDA) receptor-gated ion channels. Alcohol has opposite effects on the NMDA receptor, depending on the duration of exposure. Acute exposure to alcohol inhibits ion flow through NMDA receptors, whereas chronic exposure upregulates the number of these receptors and thereby increases ion flow. Acute withdrawal from alcohol results in hyperexcitability and seizures in the presence of upregulated NMDA receptors, making postsynaptic neurons vulnerable to excitotoxic damage. For this study, 13 grossly and histologically normal brains from alcoholics and 13 brains from nonalcoholic controls were selected from our brain bank. The two groups were matched for age, postmortem interval, and storage time. Maximal binding and affinities of NMDA receptors were determined by quantitative autoradiography in the cingulate cortex, the cornu Ammonis of the hippocampus, and in the cerebellar vermis. Binding was determined with an agonist, L-[3H]glutamate, with a competitive antagonist, [3H]CGP-39653, and with an antagonist binding in the channel interior, [3H]MK-801. No significant differences were found in receptor densities or affinities between alcoholics and controls. Real differences were not likely to be obscured by nonalcohol-related variables because the groups were closely matched for age, autopsy delay, time in storage, and central nervous system medications. Various diseases causing acute and chronic hypoxia did not significantly affect receptor density or affinity. Liver diseases and thiamine deficiency were excluded. A long-lasting upregulation of the number or affinity of NMDA receptors is not a key feature of chronic alcoholics.

NMDA-receptor activity visualized with (S)-[N-methyl-11C]ketamine and positron emission tomography in patients with medial temporal lobe epilepsy.

To determine whether neurochemical activation of the N-methyl-D-aspartate (NMDA) receptor-gated ion channel shows quantitative changes, measured as binding of 11C-labeled (S)-[N-methyl]ketamine, in patients with medial temporal lobe epilepsy (MTLE). Eight patients with MTLE who were evaluated regarding epilepsy surgery underwent positron emission tomography (PET) with (S)-[N-methyl-11C]ketamine. The presurgical investigations included magnetic resonance imaging (MRI), PET with 18F-fluoro-deoxyglucose (18FDG), and seizure monitoring by using video-EEG. The uptake of (S)-[N-methyl-11C]ketamine in the temporal lobe of ictal onset was compared with the contralateral side and correlated to changes in regional glucose metabolism measured by PET with 18FDG. (S)-[N-methyl-11C]ketamine rapidly reached the brain, and high radioactivities were measured in the striatum, thalamic nuclei, and cortical regions. Overall the brain uptake and regional binding potentials of (S)-[N-methyl-11C]ketamine were similar to measurements observed previously in healthy controls. However, 20 min after administration, when blood flow influence was negligible, a side-to-side comparison revealed a 9-34% reduction of tracer radioactivity in the temporal lobes of ictal onset. At earlier times, the differences in binding potentials were less pronounced, 9-21%. The magnitude and distribution of the reduction were similar to the metabolic pattern seen on PET scans with 18FDG. Radioactivity uptake of intravenously administered (S)-[N-methyl-11C]ketamine was reduced in temporal lobes of ictal in patients with TLE. This may reflect reduced NMDA-receptor density, reduced perfusion, focal atrophy, or other factors.

Glutamate receptors in the frontal cortex of alcoholics.

This study tests the hypothesis that glutamate receptors are altered in the brains of alcoholics as a result of chronic alcohol neurotoxicity. Release of the neurotransmitter glutamate after seizures or brain ischemia may damage postsynaptic neurons by increasing calcium flux through N-methyl-D-aspartate (NMDA) receptor-gated ion channels. Alcohol has two opposite effects on glutamate receptor ion channel complexes, depending upon the duration of exposure. Acute exposure to alcohol inhibits ion flow through these receptor-channel complexes, whereas chronic exposure up-regulates the number of these receptors and thereby increases ion flow. Acute withdrawal from alcohol results in hyperexcitability and seizures in the presence of up-regulated channels, thereby making postsynaptic neurons vulnerable to excitotoxic damage. We selected 13 histologically normal brains from alcoholics and 13 brains from controls from our brain bank that were matched for age, postmortem interval, and storage time. Maximal binding and affinities of glutamate receptor subtypes were determined by quantitative autoradiography in the superior frontal cortex, Brodmann area 8. The most alcohol-sensitive subtype, NMDA receptor-channel complexes, were modestly but consistently increased in alcoholics. This included agonist sites (NMDA-sensitive [3H]glutamate), and antagonist site ([3H]CGP-39653), and a [3H]MK-801 binding site in the channel interior, although the increase of the latter did not reach statistical significance. Age, autopsy delay, time in storage, liver diseases, thiamine deficiency, CNS medications, and various diseases causing acute and chronic hypoxia did not significantly affect receptor density or affinity. In contrast, the other two glutamate channel subtypes, AMPA and kainate receptors, were not significantly different in alcoholics compared with controls. In conclusion, chronic alcoholism moderately increases the density of the NMDA subtype of glutamate receptors in the frontal cortex. This up-regulation may represent a stage of alcohol-induced chronic neurotoxicity.

MK-801 reduces uptake and stimulates efflux of excitatory amino acids via membrane depolarization.

MK-801 and related compounds reduce excitotoxic neuronal injury by blocking N-methyl-D-aspartate (NMDA) receptorgated ion channels. These agents also cause neuronal vacuolization and block glutamate-induced astrocyte swelling, effects that may be unrelated to actions at the NMDA receptor. In the present study, high concentrations of MK-801 (100-1,000 microM) caused uncompetitive inhibition of glutamate uptake in astrocyte and neuronal cultures and stimulated D-aspartate efflux from astrocytes. MK-801 (500 microM) reduced the maximal velocity for glutamate uptake in astrocytes from 31 to 17 nmol.mg protein-1.min-1, whereas competitive NMDA receptor antagonists did not affect glutamate uptake. MK-801 also inhibited uptake of gamma-aminobutyric acid (GABA). Because both GABA uptake and glutamate uptake are electrogenic, one mechanism by which MK-801 could inhibit uptake is by membrane depolarization. Whole cell patch-clamp recording confirmed that MK-801 in the range of 100-1,000 microM caused dose-dependent and reversible depolarization. These concentrations are far higher than necessary to block NMDA receptors, and the findings suggest that actions at sites other than NMDA receptors could contribute to the effects of high doses of MK-801 in some experimental and clinical settings.